TLC Tips & Tricks |
Avoid Errors and Simplify Your Work with Our Practical TLC Tips
Discover TLC tips and tricks for the following topics:
1. Choice of Solvent System (Mobile Phase)
2. Choice of TLC Layer (Stationary Phase)
3. Pre-Conditioning TLC Plates
4. Correct sample application
5. Drying TLC Plates
6. How to Saturate TLC Chambers
7. Spraying TLC Plates for Derivatization
8. Quantitative Evaluation with TLC Scanners
TLC Tip 1: Choice of Solvent System (Mobile Phase)
The choice of solvent system is critical in thin-layer chromatography. Follow the guidelines and table below to find the most suitable mobile phase for your separation.
- To choose the right solvent, start with pure solvents of medium elution strength.
- Perform spot tests to compare different solvent systems.
- Single solvents are seldom used in TLC; most solvent systems contain several components, but keep it as simple as possible.
- The solvent system must be capable of wetting the TLC layer.
- Use appropriate solvent purity
- Refer to scientific literature or pharmacopoeia monographs to facilitate your search.
In the table below, the solvents are listed in increasing order of elution strength (according to Halpaap’s eluotropic series).
| Solvent | Velocity coefficient, k (mm²/s) | ||
|---|---|---|---|
Lower elution strength ↓ Higher elution strength |
1 | n-Heptane | 11.4 |
| 2 | n-Hexane | 14.6 | |
| 3 | n-Pentane | 13.9 | |
| 4 | Cyclohexane | 6.7 | |
| 5 | Toluene | 11.0 | |
| 6 | Chloroform | 11.6 | |
| 7 | Dichloromethane | 13.2 | |
| 8 | Diisopropyl ether | 13.2 | |
| 9 | tert-Butanol | 1.1 | |
| 10 | Diethyl ether | 15.3 | |
| 11 | Isobutanol | 1.6 | |
| 12 | Acetonitrile | 15.4 | |
| 13 | Isobutyl methyl ketone | 9.1 | |
| 14 | 2-Propanol | 2.5 | |
| 15 | Ethyl acetate | 12.1 | |
| 16 | 1-Propanol | 2.9 | |
| 17 | Ethyl methyl ketone | 13.9 | |
| 18 | Acetone | 16.2 | |
| 19 | Ethanol | 4.2 | |
| 20 | 1,4-Dioxane | 6.5 | |
| 21 | Tetrahydrofuran | 12.6 | |
| 22 | Methanol | 7.1 | |
| 23 | Pyridine | 8.0 | |
| Sorbent | TLC plate silica gel 60 F254 Merck | ||
| Type of chamber | N-chamber with chamber saturation | ||
| Room temperature | 22 °C | ||
| Migration distance of solvent | 100 mm | ||
| Source: Applied Thin-Layer Chromatography, Elke Hahn-Deinstrop, page 71 | |||
TLC Tip 2: Choice of TLC Layer (Stationary Phase)
To help you select the optimal stationary phase for your analysis, the table below shows the most popular pre-coated TLC layers available and their typical applications.
| Sorbent material | Chromatographic principle | Typical applications |
|---|---|---|
| Aluminum oxide |
Adsorption chromatography due to polar interactions |
Alkaloids, steroids, terpenes, aliphatic, aromatic and basic compounds |
| Cellulose | Depending on acetyl content transition from normal phase to reversed phase chromatography | Anthraquinones, antioxidants, polycyclic aromatics, carboxylic acids, nitrophenols, sweeteners |
| Kieselguhr | Commonly impregnated for reversed phase separations | Aflatoxins, herbicides, tetracyclines |
| Silica | ||
| Standard silica gel, also with concentrating zone | Normal phase chromatography | Most frequent application of all TLC layers, Aflatoxins |
| Silica gel G, impregnated with ammonium sulfate | Surfactants, lipids (neonatal respiratory syndrome) | |
| Silica gel 60, impregnated with caffeine for PAH determination | Charge transfer complexes | Polycyclic aromatic hydrocarbons (PAH) acc. to German drinking water specification |
| Cyano-modified layer CN | Normal phase and reversed phase chromatography | Pesticides, phenols, preservatives, steroids |
| DIOL-modified layer | Steroids, hormones | |
| Amino-modified layer NH2 |
Anion exchange, normal phase and reversed phase chromatography |
Nucleotides, pesticides, phenols, purine derivates, steroids, vitamins, sulfonic acids, carboxylic acids, xanthines |
| RP layers | ||
| RP-2, RP-8, RP-18 | Nonpolar substances (lipids, aromatics) | |
| Silica gel 60 silanized | Polar substances (basic and acidic pharmaceutical active ingredients) | |
| RP-18 W/UV254, wettable | Normal phase and reversed phase chromatography | Aminophenols, barbiturates, preservatives, nucleobases, PAH, steroids, tetracyclines, phthalates |
| Spherical silica gel | ||
| LiChrospher® Si 60 | Normal phase chromatography | Pesticides, phytopharmaceuticals |
| Source: Applied Thin-Layer Chromatography, Elke Hahn-Deinstrop, pages 22-23 | ||
TLC Tip 3: Pre-Conditioning TLC Plates
Pre-conditioning TLC layers protects them from humidity, which could otherwise diminish their activity and affect chromatogram results.
- A common pre-conditioning method is to place the TLC plate in a development chamber containing highly saturated salt solution with a large amount of undissolved salt, and allowing the plate to condition for several hours. For reproducible results, make sure the solution contains sufficient undissolved salt!
- Other pre-conditioning methods include modifying the TLC layer by exposure to gas, or conditioning the plate with organic solvents, acids or bases.
- During sample application, cover the application area with a clean glass plate to maintain the layer’s activity until development is completed.
TLC Tip 4: Correct sample application
The correct sample application on TLC plates is essential for accurate and reproducible separations. Below are a few ways you can avoid errors.
- Record the position of each sample on the data sheet.
- Cross out used lanes to prevent repeated application on any lane, and to ensure that no samples are omitted.
- Avoid applying samples too close to the plate’s edge or to the solvent surface.
- Leave sufficient space between application areas.
- Ensure a consistent distance from the bottom edge of the plate for all samples.
Learn more about sample application
TLC Tip 5: Drying TLC Plates
Highly volatile compounds (e.g. α-pinene)
- Dry plates in a cool room to avoid sample evaporation prior to development.
Volatile compounds (e.g. essential oils applied with toluene or n-hexane)
- Dry plates horizontally for a few minutes at room temperature before placing them in the development chamber.
Thermally stable substances (up to 1000 µg/lane from chloroform or methanol)
- Apply uniform heat at a temperature close to the solvent’s boiling point for around 20 minutes.
Thermally labile or oxidation-prone samples
- Carry out several drying tests prior to separation.
Important: Keep exposure of plates to blowers as short as possible to protect the layer from airborne dirt particles.
TLC Tip 6: How to Saturate TLC Chambers
TLC development can be performed in saturated or unsaturated chambers. Chromatography in unsaturated chambers results in evaporation of the solvent from the layer, particularly near the front. This leads to higher solvent consumption, and higher Rf values.
Chamber saturation method
- Line the chamber with strips of filter paper, leaving a gap for observation.
- Fill the chamber with solvent to a height of 0.5 to 1 cm.
- Carefully tilt the chamber to moisten the filter paper and equilibrate the chamber with solvent vapors. After a few minutes, the chamber is saturated with vapors.
- Place the TLC plate in the chamber carefully so that the solvent does not spill over the starting line. Contact between the side of the plate and the filter paper must also be avoided.
- Development can now proceed.
Learn more about TLC development
TLC Tip 7: Spraying TLC Plates for Derivatization
Safety
- Airborne solvents may be toxic. Wear goggles, gloves and a dust mask while spraying, and ensure good ventilation.
- Avoid chlorinated hydrocarbons (CHC’s) to protect yourself and the environment.
Challenges
- Spraying produces a less uniform coating than dipping or in-situ derivatization.
- Difficult to control reagent quantity while spraying.
Recommendations
- Always use fresh reagents for each application.
- Reagents stored for long periods should be thoroughly tested prior to usage.
Learn more about TLC derivatization
TLC Tip 8: Quantitative Evaluation with TLC Scanners
- Ensure that all chromatograph lanes are complete before placing the plate in the TLC scanner.
- For accurate analysis of complex sample mixtures, apply the sample as a band (instead of a spot).
- To establish the detection limit, use a blank lane outside the sample lanes for comparison.
- To avoid difficulties with linearity, keep the sample concentration range at a moderate limit.